EP1728079A1 - Methodes et puces a adn pour la detection de petits composes moleculaires - Google Patents

Methodes et puces a adn pour la detection de petits composes moleculaires

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Publication number
EP1728079A1
EP1728079A1 EP05732959A EP05732959A EP1728079A1 EP 1728079 A1 EP1728079 A1 EP 1728079A1 EP 05732959 A EP05732959 A EP 05732959A EP 05732959 A EP05732959 A EP 05732959A EP 1728079 A1 EP1728079 A1 EP 1728079A1
Authority
EP
European Patent Office
Prior art keywords
biochip
small molecule
binding
control
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP05732959A
Other languages
German (de)
English (en)
Other versions
EP1728079A4 (fr
Inventor
Yimin Sun
Wanli Xing
Guoqing Wang
Hongwu Du
Rong Zhang
Yuan Chen
Yan Wang
Jing Cheng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tsinghua University
CapitalBio Corp
Original Assignee
Tsinghua University
CapitalBio Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tsinghua University, CapitalBio Corp filed Critical Tsinghua University
Publication of EP1728079A1 publication Critical patent/EP1728079A1/fr
Publication of EP1728079A4 publication Critical patent/EP1728079A4/fr
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00387—Applications using probes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/0061—The surface being organic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates methods of detecting compounds and devices for detecting compounds. More particularly, the present invention relates to methods of detecting small molecule compounds and biochips for detecting small molecule compounds.
  • Biochip technology is one of the most important advancements in science and technology since mid-nineties. It is a technology crossing biology, electronics, physics, chemistry and computer science. Biochip technology generally includes the following procedures: first, biochips are generated by immobilizing biological molecules such as nucleic acid fragments, peptides and even cells and tissues in some. order onto a solid support such as glass slide, silica slide, hydrogel and membrane; the biochip generated is reacted with target molecules in samples; finally, the signal intensities of the biochip are analyzed effectively through the special apparatus such as scanner to analyze the concentration of the target molecules in the sample.
  • a solid support such as glass slide, silica slide, hydrogel and membrane
  • biochips Based on the differences of the immobilized molecules, biochips can be classified into gene microarray, protein microarray, cell microarray and tissue microarray. Lab-on-chip developed in recent years is also an important branch of biochip technology.
  • the two main methods currently used for small molecule detection are physical analysis and immunological analysis. Physical analysis mainly includes spectrum method, chromatography, and combination of these methods. Chromatography detection is mostly used, such as HPLC, GC and TLC. Immunological analysis includes RIA, ELISA, FIA, among which ELISA is mostly used.
  • Chromatography separation system mainly includes the separated components, fluid phase and stationary phase. The separation principle is based on the distribution coefficie ⁇ cy difference of each component in the two phases.
  • ELISA separation When the two phases move relative to each other, the components are separated by distributing repeatedly between the two phases along the movement of the fluid phase. Chromatography separation has the advantages of high effectiveness, good selectivity and accurate qualitative and quantitative analysis. But it also has some disadvantages such as complicate sample preparation, expensive apparatus and long time detection period.
  • Immunological analysis such as ELISA of small molecules is a kind of combination technology of immunology, analytical chemistry and synthetic chemistry. There are two ways of using ELISA for detection of small molecules. One way is that the antibody is immobilized and the detection is completed by enzyme-linked small molecule. The other way is that the carrier-linked small molecule is immobilized and the detection is completed by enzyme-linked antibody. ELISA has the advantages of high sensitivity, low detection cost and short time detection period. However, its disadvantage is single-target detection.
  • the present invention provides methods and biochips for detecting small molecule compounds.
  • the invention provides a biochip for detecting a small molecule compound comprising a solid support and a conjugate of a carrier and a small molecule compound, wherein the conjugate is immobilized on a surface of the solid support.
  • the small molecule compound has a molecular weight ranging from 1 to 10,000 daltons.
  • the small molecule compound is a veterinary drug selected from the group consisting of enrofloxacin, furantoin, furacilin, furazolidone, ciprofloxacin, sulfadimidine, sulfamethoxydiazine, sulfamethazine, sulfadimoxinum, sulfamethoxazole, sulfamerazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfaquinoxaline, sulfadiazine, sulfathiazole, chlortetracycyline, clenbuterol, streptomycin, chloramphenicol, norfloxacin, difloxacin, dihydrostreptomycin, tetracycline, oxytetracycyline, digoxin, aflatoxins, kanamycin, mercaptoethanol, pen
  • the small molecule compound is a prohibited substance selected from the group consisting of amphetamine, benzoylecgonine, phencyclindine, theophylline, barbiturate methadone, benzodizepine, morphine, tricyclic antidepressant, gentamicin, digoxin, estradiol, tobramycin.
  • the carrier is a protein selected from the group consisting of human serum albumin (HSA), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), and ovabumin (OVA).
  • HSA human serum albumin
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • OVA ovabumin
  • a plurality of conjugates are immobilized on the solid support to form a two-dimensional array.
  • the biochip further comprises one or more control immobilized on the surface of the solid support, wherein the control is selected from the group consisting of a blank control, a negative control, a sample preparation control, an immobilization control, and a data normalization control.
  • the biochip comprises a blank control, a negative control, a sample preparation control, an immobilization control, and a data normalization control immobilized on the surface of the solid support.
  • the solid support is selected from the group consisting of ceramic, glass, silica, quartz, nylon, plastic, polystyrene, nitrocellulose, and metal.
  • the invention also provides a method of making a biochip for detecting a small molecule compound, said method comprising: a) linking a small molecule compound to be detected to a carrier to form a conjugate; b) spotting the conjugate onto a chemically modified surface of a solid support; and c) drying the spotted solid support.
  • the invention also provides a method for detecting a small molecule compound in a sample, said method comprising: a) incubating a biochip described herein with a sample and a binding molecule that specifically binds to the small molecule compound under conditions suitable for specific binding of the binding molecule to the small molecule compound; b) detecting binding of the binding molecule to the small molecule compound in the conjugate immobilized on the surface of the biochip, whereby the presence or absence or the quantity of the small molecule compound in the sample is detected.
  • the biochip is incubated in a blocking solution before step a).
  • the biochip in step a) is incubated with a mixture of the sample and the binding molecule.
  • the biochip in step a) is first incubated with the sample and then incubated with the binding molecule. In some embodiments, the biochip in step a) is first incubated with the binding molecule and then incubated with the sample. In some embodiments, the method further comprises a step of comparing the binding of the binding molecule to the small molecule compound in the conjugate immobilized on the surface of the biochip to binding of the binding molecule to a control immobilized on the surface of the biochip. In some embodiments, the binding molecule is an antibody or a polymer.
  • the binding molecule is linked to a label, and binding of the binding molecule to the small molecule compound in the conjugate immobilized on the surface of the biochip is detected by detecting the presence or absence or quantity of the label on the biochip.
  • the label may be a molecule selected from the group consisting of a fluorescent, an enzymatic, a biotin, a radioactive, and a luminescent label.
  • the method further comprises a step of incubating the biochip with a secondary antibody that specifically binds to the binding molecule, and the binding of the binding molecule to the small molecule compound in the conjugate immobilized on the surface of the biochip is detected by detecting binding of the secondary antibody.
  • the secondary antibody is linked to a label, and binding of the secondary antibody to the binding molecule is detected by detecting the presence or absence or quantity of the label on the biochip.
  • the method of the invention is used for detecting residual veterinary drug or detecting abuse of prohibited substances.
  • the invention also provides a kit for use in any of the detection methods described herein.
  • the kit comprises a biochip described herein and a binding molecule that specifically binds to the small molecule compound.
  • the kit may further comprise instructions for use of detecting small molecule compounds described herein.
  • Figure 1 shows a biochip reacted with a negative sample in which the residual sulfadimidine, streptomycin and enrofloxacin were lower than the maximum residue limit
  • FIG. 1 shows a biochip reacted with a positive sample in which the residual enrofloxacin was higher than the MRL, but the residual sulfadimidine and streptomycin were lower than the MRL.
  • Figure 3 shows a biochip reacted with a positive sample in which the residual sulfadimidine was higher than the MRL, but the residual enrofloxacin and streptomycin were lower than the MRL.
  • Figure 4 shows a biochip reacted with a positive sample in which the residual streptomycin was higher than the MRL, but the residual enrofloxacin and sulfadimidine were lower than the MRL.
  • Figures 1-4 all the positive drugs are labeled with white frame and other spots are the negative drugs and controls which can improve the reliability of results.
  • Figure 5 shows a typical appearance and layout of the arrays.
  • A Schematic diagram showing the glass slide and the polyester framing the reaction chambers above each array.
  • B Drug layout on the array (each drug/substance is printed in triplicate).
  • C Image of one of the 9 x 9 array.
  • PCP phencyclindine
  • TCA tricyclic antidepressants
  • hCG human chorionic gonadotropin
  • LH luteinizing hormone.
  • Figure 6 shows result of analysis for amphetamine on a chip. The gray box indicates the expected binding of an antibody to its counterpart.
  • Figure 7 is a graph showing calibration curve for amphetamine measured with the biochip. Calibration curve was produced by using different concentrations (0-1024 ug/L) of each substance added to a drug-free urine.
  • FLU fluorescence.
  • the present invention provides methods for detecting small molecule compounds using biochips.
  • the present invention has advantages of both biochip technology and immunological analysis. For example, multi-sample detection may be performed at the same time on one biochip. Because of the biochip technology, many targets may be analyzed simultaneously in only one cycle of detection. The results are more reliable. Every step of the detection cycle may also be effectively controlled by the controls in the biochip to confirm the reliability of the results. Small volume of samples are required. About ten- microlitre sample may be enough for a cycle of detection.
  • the present invention provides many advantages, such as high throughput and abundant information from biochip technology, and simple operation, fast detection, high sensitivity and low cost from immunological analysis.
  • sample refers to anything which may contain a target small molecule compound that may be assayed by the present methods, kits and chips.
  • the sample may be a biological sample, such as a biological fluid or a biological tissue.
  • biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.
  • Biological tissues are aggregates of cells, usually of a particular kind together with their intercellular substance that form one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelium, muscle and nerve tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s). Biological tissues may be processed to obtain cell suspension samples. The sample may also be a mixture of cells prepared in vitro. The sample may also be a cultured cell suspension.
  • the sample may be crude samples or processed samples that are obtained after various processing or preparation on the original samples.
  • various cell separation methods e.g., magnetically activated cell sorting
  • chip biological chip
  • microarray chip refers to a solid substrate with a plurality of one-, two- or three-dimensional micro structures or micro-scale structures on which certain processes, such as physical, chemical, biological, biophysical or biochemical processes, etc., can be carried out.
  • the micro structures or micro-scale structures such as, channels and wells, can be incorporated into, fabricated on or otherwise attached to the substrate for facilitating physical, biophysical, biological, biochemical, chemical reactions or processes on the chip.
  • the chip may be thin in one dimension and may have various shapes in other dimensions, for example, a rectangle, a circle, an ellipse, or other irregular shapes.
  • the size of the major surface of chips, upon which the processes can be carried out, can vary considerably, e.g., from about 1 mm2 to about 0.25 m2.
  • the size of the chips is from about 4 mm2 to about 25 cm2 with a characteristic dimension from about 1 mm to about 5 cm.
  • the chip surfaces may be flat, or not flat.
  • an "antibody” (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target, such as a small molecule compound, carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
  • An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class.
  • Biochips for detecting small molecule compounds The present invention provides a biochip for detecting a small molecule compound comprising a solid support and a conjugate of a carrier and a small molecule compound, wherein the conjugate is immobilized on a surface of the solid support.
  • the biochip comprises a solid support and one type of conjugate.
  • the biochip comprises a solid support and a plurality of different conjugates.
  • the invention also provides a method of making a biochip for detecting a small molecule compound, said method comprising: a) linking a small molecule compound to be detected to a carrier to form a conjugate; b) spotting the conjugate onto a chemically modified surface of a solid support; and c) drying the spotted solid support.
  • Any small molecule compounds that can be conjugated to a carrier and specifically bind to a binding molecule may be detected using the methods and biochips of the present invention.
  • the small molecule compound of the present invention may have a molecular weight ranging from about 1 to about 10,000, from about 100 to about 5,000, from about 200 to about 2,000 daltons. In some embodiments, the small molecule compound is a veterinary drug.
  • Exemplary veterinary drugs include, but are not limited to, enrofloxacin, furantoin, furacilin, furazolidone, ciprofloxacin, sulfadimidine, sulfamethoxydiazine, sulfamethazine, sulfadimoxinum, sulfamethoxazole, sulfamerazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfaquinoxaline, sulfadiazine, sulfathiazole, chlortetracycyline, clenbuterol, streptomycin, chloramphenicol, norfloxacin, difloxacin, dihydrostreptomycin, tetracycline, oxytetracycyline, digoxin, aflatoxins, kanamycin, mercaptoethanol, penicillins, gentamicin,
  • any one or more of these veterinary drugs may be conjugated to a carrier, and any combination of the conjugate may be immobilized onto a solid support of a biochip.
  • Other small molecule compounds that can be detected using the biochip described herein includes stimulants, narcotics, anabolic agents, and peptide hormones. In some embodiments, the small molecule compound is a prohibited substance.
  • Exemplary prohibited substances include, but are not limited to, amphetamine, benzoylecgonine, phencyclindine, theophylline, barbiturate methadone, benzodizepine, morphine, tricyclic antidepressant, gentamicin, digoxin, estradiol, tobramycin, amineptine, amiphenazole, bromantan, caffeine, carphedon, cocaine, ephedrines, fencamfamine, mesocarb, pentylentetrazol, pipradol, salbutamol, salmeterol, terbutaline, dextromoramide, diamorphine (heroin), methadone, morphine, pentazocine, pethidine, rostenedione, clostebol, dehydroepiandrosterone (DHEA), fluoxymesterone, metandienone, nandrolone, oxandrolone, stanozolol, testosteronectenbut
  • any one or more of these prohibited substances may be conjugated to a carrier, and any combination of the conjugates may be immobilized onto a solid support of a biochip.
  • the small molecules compound is conjugated to a carrier before being immobilized on the biochip.
  • the small molecule compound may be coupled or linked to the carrier in any ways known in the art.
  • the small molecule compound is cross-linked to the carrier using one or more crosslinking agents via functional groups on the small molecule compound and the carrier.
  • the functional group on the small molecule and/or the carrier may be modified in order to react with a specific cross-linking agent.
  • Cross-linking agents that may be used include, but not limited to, dicyclohexylcarbodi-imide (DCC), N- hydroxy-succinimide (NHS), l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters, disuccinimidyl esters, 3,3'-dithiobis(succinimidylpropionate)), and bifunctional maleimides (e.g.,bis-N-maleimido-l,8-octane).
  • DCC dicyclohexylcarbodi-imide
  • NHS N- hydroxy-succinimide
  • NHS N- hydroxy-succinimide
  • l,l-bis(diazoacetyl)-2-phenylethane glutaraldehyde
  • the small molecule compound and the carrier are linked via biotin-streptavidin or biotin-avidin interaction.
  • the small molecule compound may be biotinalized and the carrier protein is linked to a strepavidin molecule.
  • Other methods known in the art can be used to conjugate the small molecule compound to the carrier.
  • the small molecule compounds are conjugated to carriers before being immobilized onto a biochip.
  • Carriers can be any molecules that are useful for immobilizing the small molecule compounds onto a solid support and presenting the small molecule compounds to a binding molecule.
  • Exemplary carriers include, but are not limited to, proteins, polypeptides, polymers, nucleic acids.
  • SA serum albumin
  • HAS human serum albumin
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • OVA ovabumin
  • the conjugates may be immobilized onto a surface of a biochip using any methods known in the art.
  • the surface of the biochip may be chemically modified, such as glass slides modified with aldehyde groups.
  • Example 3 describes methods of immobilizing conjugates onto aldehyde-activated glass slides.
  • the conjugates may be spotted onto the surface using any techniques known in the art, such as automated spotting apparatus.
  • the biochip may be dried to allow immobilization of the conjugates onto the surface of the biochip.
  • the biochip may also have one or more controls immobilized on the same surface as the conjugates. Exemplary controls are blank controls, negative controls, sample preparation controls, immobilization controls, and data normalization controls.
  • One or more conjugates of the small molecule compounds and the carriers may be immobilized onto the biochips to form a two-dimension array, for example, a 9 x 9 array, 12 x 12 array, and 15 x 15 array.
  • One or more arrays may be arranged on one biochip, and one or more samples can be tested using one biochip.
  • the sample volume used for testing may be less than about any of 1 ml, 0.5 ml, 0.25 ml., 0.1 ml, 0.05 ml, and 0.01 ml.
  • the solid support of the biochip comprises a surface selected from the group consisting of a ceramic, a glass, a silica, a quartz, a nylon, a plastic, a polystyrene, a nitrocellulose, and a metal.
  • the present invention uses a competitive immunoassay.
  • the invention provides a method for detecting a small molecule compound in a sample, said method comprising: a) incubating a biochip described herein with a sample and a binding molecule that specifically binds to the small molecule compound under conditions suitable for specific binding of the binding molecule to the small molecule compound; b) detecting binding of the binding molecule to the small molecule compound in the conjugate immobilized on the surface of the biochip, whereby the presence or absence or the quantity of the small molecule compound in the sample is detected.
  • the biochip may be first incubated in a blocking solution for blocking nonspecific binding, for example, blocking the non-spotted area on the biochip.
  • Any blocking solution used for immunoassay may be used.
  • PBS phosphate-buffered saline
  • BSA phosphate-buffered saline
  • the biochip may be washed before the next step.
  • the biochip may be incubated with a mixture of the sample to be tested and the binding molecule.
  • the biochip may also be incubated first with the sample and followed by incubation with the binding molecule, or incubated first with the binding molecule and followed by incubation with the sample.
  • Any binding molecule that specifically binds to the small molecule compound may be used, for example, antibodies, polypeptides, and polymers.
  • a binding molecule specifically binds to an epitope or a small molecule compound is a term well understood in the art, and methods to determine such specific binding are also well known in the art.
  • a molecule is said to exhibit "specific binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular substance than it does with alternative substances. Since different molecules may have the same or similar epitope, a binding molecule may cross-react with more than one compounds.
  • Specific binding used herein includes specific binding to an epitope or structurally related compounds.
  • Binding molecules that cross-react with more than one structurally related small molecules may be used to detect these small molecules, and the binding of such a binding molecule to the biochip may indicate the presence and/or quantity of any of these small molecules that the binding molecule cross-reacts with. After the incubation, the biochip may be washed before binding detection. Binding of the binding molecules to the small molecules in the conjugates immobilized on the biochips can be detected using any methods known in the art.
  • the binding molecules (such as, antibodies and polymers) are linked to a label, such as , a fluorescence, an enzyme, a biotin, a radioisotope, and a luminescence.
  • the binding of the binding molecules to the biochip are detected by detecting the presence or absence, and/or quantity of the label on the biochip. Any labels and methods known in the art for detecting the labels may be used. Since this is a competitive immunoassay design, the absence or lower level of the signal indicates the presence and higher quantity of the small molecule compound in the sample tested. Binding of the binding molecule to the biochip may also be detected using a secondary binding molecule which is linked to a label. Any label known in the art and described herein may be used. After incubation with the binding molecule and the sample, the biochip is further incubated with the secondary binding molecule which specifically binds to the binding molecule. In some embodiments, the secondary binding molecule is an antibody.
  • Kits for detecting small molecule compounds The present invention also provides a kit for detecting a small molecule compound in a sample, said kit comprising one or more biochips described herein and one or more binding molecules that specifically bind to the small molecule compounds.
  • the kits may include one or more containers and may further comprise instructions for use in accordance with any of the methods described herein.
  • the instructions supplied in the kits are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine -readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.
  • the label or packaging insert may indicate that the biochip and the binding molecule are used for detecting small molecule compounds, such as veterinary drugs, or prohibited substances.
  • kits of this invention may be in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components, such as blocking solution and washing solution, control samples, buffers and interpretive information.
  • Example 1 Preparation of biochips for residual veterinary drugs detection
  • the biochips were prepared in four steps as described below. Step 1. Spotting solutions were prepared by dissolving each of BSA-linked enrofloxacin, OVA-linked sulfadimidine, OVA-linked streptomycin, negative control, sample preparation control, immobilization control, and data normalization control in spotting buffer (40% glycerol, 60% PBS) at protein concentration of 1.0 mg/mL. Each potting solution was then transferred into the 384-hole plate for spotting onto a biochip.
  • spotting buffer 50% glycerol, 60% PBS
  • enrofloxacin and BSA conjugated to the carrier proteins as described below: Conjugation of enrofloxacin and BSA: 1) 1200 mg hydrochloric enrofloxacin was added into 1.0 ml pure water. The pH of solution was adjusted to pH 6.0 with 2 mol/L NaOH. The solution was incubated at 4°C for 30 minutes. Then dicyclohexylcarbodi-imide (DCC) and N-hydroxy-succinimide (NHS) (both from Sigma) solution were added, and reaction was allowed for 30 minutes. 2) l.Og BSA was added into 0.2 mol/L phosphate buffer (pH 7.2), mixed.
  • DCC dicyclohexylcarbodi-imide
  • NHS N-hydroxy-succinimide
  • the BSA solution was then slowly added into the solution prepared in step 1), and the mixed solution was incubated at 4°C overnight to form the conjugate.
  • the BSA and enrofloxacin solution prepared in step 2) was dialyzed against phosphate buffer for 5 days. The phosphate buffer was changed at least 12 times. The dialyzed conjugate solution was stored at -20°C.
  • the conjugate solution was stored at - 20°C. Step 2.
  • the above spotting solutions were distributed in some order onto the chemically modified glass chips by automated spotting apparatus. Each chip included 10 arrays (5 rows X 2 columns) and each array included 36 sample spots (6 rows X 6 columns) in which the interval between two spots was 400 um. Each array was an isolated reaction chamber. Step 3. After spotting, the chips were dried with vacuum machine. Step 4. Once dried, the chips were vacuum-packed and stored at 4°C. Chips prepared as described above can be used to detect enrofloxacin, sulfimidine and streptomycin in qualitative analysis, semi-quantitative analysis and quantitative analysis.
  • Example 2 Detection of residual veterinary drug with biochips. Samples containing residual enrofloxacin, sulfimidine or streptomycin were tested as described below: 1. Blocking: The biochip prepared as described in Example 1 was blocked with 10% goat serum in 37°C for 30 minutes. 2. Cleaning and drying: The biochip was then washed in the washing cassette with PBST (PBS containing 0.5% Tween-20) for 5 minutes with agitation, then was centrifuged in 1000 rpm for 1 min in order to dry the chip. 3.
  • PBST PBS containing 0.5% Tween-20
  • the first antibody reaction The sample to be tested was mixed with an antibody that specifically binds to enrofloxacin, an antibody that specifically binds to sulfimidine, and an antibody that specifically binds to streptomycin (anti-streptomycin antibody was obtained from Beijing Wanger Biotech, Ltd.) with each antibody at 1 mg/ml concentration. Twenty ul of the mixture of the sample and antibodies were added into the reaction and reaction was allowed for 30 min at 37°C. 4.
  • the second antibody reaction The chip was washed and dried as described in step 2. Then, 20 ul goat-anti-mouse IgG labeled with fluorescence was added into the reaction chamber at a concentration of 1 ug/ml. The chip was incubated at 37°C for 30 minutes.
  • Chip scan and data analysis The chip was then washed and dried as described in step 2. The chip was then scanned and the data were analyzed. The results are shown in Figures 1-4. Since competitive immunoassay was used, the lower signal spot indicates higher level of residual veterinary drug present in the sample tested.
  • the sensitivity of the detection system for detecting small molecule compounds of the present invention meets the technical target and maximum residual level (MRL) allowed by Chinese government. The sensitivity and linear range was compared to the MRL in Table 1 below.
  • MRL showed in the table was the minimum of MRLs for various type of samples.
  • the sample to be tested may be diluted because the sensitivity of system is much higher than the MRL.
  • concentrations of residual veterinary drugs in Figures 1-4 are shown in Table 2 below. Table 2. The residual veterinary drugs in Figures 1-4.
  • Example 3 Detection of prohibited substances with biochips Sample collection. Urine samples were collected and stored at -20°C. Positive control and negative control urine samples were also collected. Details of sample collection are described in Du et al., Clinical Chemistry 51:368-375 (2005). Preparation of chip substrates. Glass slides chemically modified with aldehyde groups were used as the substrates to covalently bind BSA-co ⁇ jugated molecules at the designated locations. The slides were cleaned with 100 g/L chromic acid for 6 h, followed by rinsing with deionized water.
  • slides were then dipped into a 2 mol/L sodium hydroxide solution and then 4 mol/L hydrochloric acid, each for 30 min, followed by rinsing with deionized water and then drying under stream of nitrogen. Cleaned slides were silanized for 8 h using 3-glycidoxypropytrimethoxysilane in ethanol (40 mL/L). The glass surface was washed with toluene, acetone, and deionized water, after which the slides were dipped in 4 mol/L hydrochloric acid again for 30 min and then immersed into 50 mmol/L NaIO4 for 1 h to complete the preparation process.
  • the contact angles of the aldehyde-activated slides were measured by use of a contact angle system (Model OCA; DataPhysics Instruments GmbH) for quality-control purposes. Slides were stored in a desiccated box at room temperature for a maximum of 3 months. Printing of chips. Ten 9 x 9 arrays of BSA-conjugated drugs were printed on each slide. For a peptide hormone, the peptide was printed directly. On each slide, one sample can be tested on one 9 x 9 array for a variety of analytes, and up to 10 samples can be analyzed in parallel on one chip.
  • a contact printing robot (PixSys 5500; Cartesian Technologies) with a stealth microspotting pin (Model SMP3; TeleChem International) was used to print the chips on the aldehyde-activated slides.
  • concentration of each printed protein (drug-BSA) was 500 mg/L in 400 mL/L glycerol or Protein Printing Buffer (TeleChem International).
  • the drug-BSA conjugate was reacted on the chip for 6 h in a humidified chamber. The slide was then stored at room temperature for up to 1 month.
  • Immunoassay procedures A competitive immunoassay design was used to test the 16 WADA-prohibited substances on the chips.
  • a molded polyester frame was attached to the substrate to partition 10 arrays on the chip surface (Fig.
  • This chip consisted of 16 different drug-BSA conjugates and 11 positive or negative controls to form a 9 x 9 array. Each material was printed in triplicate (Fig. 5B and 5C). The chips were immersed in blocking solution (a 1:10 dilution of sheep serum in phosphate-buffered saline (PBS), pH 7.4) for 30 min at room temperature and then rinsed three times with PBS containing 0.5 mL/L Tween 20, pH 7.4 (PBS-Tween A). A mixture of the anti-drug mouse monoclonal antibodies (obtained from Fitzgerald Industries International, Inc. and Aviva Antibody Corporation) and a urine sample containing the drug was then applied to the gridded reaction chamber formed by the polyester frame covering the surface of the chip.
  • blocking solution a 1:10 dilution of sheep serum in phosphate-buffered saline (PBS), pH 7.4
  • the chip was then maintained at 37°C in a humidified chamber for 30 min.
  • the chip was then rinsed 3 times with PBS-Tween A, and the secondary antibody (Cy3-labeled goat anti-mouse IgG) was applied to the chip and incubated at 37°C for 30 min.
  • the chip was then washed again and scanned for the presence of bound Cy3 -labeled secondary antibody by use of a laser confocal scanner (GenePix 4000B; Axon Instruments) or a charge-coupled device-based scanner (EcoScan-100; CapitalBio Corporation).
  • the analog fluorescent signal was converted to digital signal by data analysis software (GenePix Pro 4.0; Axon Instruments).
  • mice IgG control spots (upper and low rows of nine spots and the two central groups of three spots) were positive, as would be expected from reaction of the immobilized mouse IgG control with the goat anti-mouse conjugate used in the assay. Influence of different matrices on the fluorescence signal on chips. The potential effect on the fluorescent signal of different samples and solutions, such as urine, water, or PBS, was evaluated. Different solutions could dramatically affect the signals for certain tested substances were found. A significant signal decrease for steroids when the solution was changed from PBS to urine was noticed. This may be attributable to the binding of some endogenous steroid interferents with the corresponding antibodies.
  • the detection limit is defined as the lowest concentration of an analyte that can be detected by the chip. This concentration corresponds to a signal that is 3 SD lower than the mean of the negative control and ranged from 0.2 ug/L for morphine to 19 ug/L for methadone.
  • the detection limit and the cutoff values (the 50% inhibitory concentration) for the 10 drugs are summarized in Table 3 below.

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Abstract

La présente invention concerne des méthodes de détection de petits composés moléculaires et de leurs puces à ADN spécifiques. Les puces à ADN de la présente invention comprennent un support solide et des petites molécules reliées à un vecteur immobilisées sur le support solide. L'invention concerne également des méthodes et des kits de détection de petits composés moléculaires au moyen des puces à ADN de l'invention.
EP05732959A 2004-03-26 2005-03-28 Methodes et puces a adn pour la detection de petits composes moleculaires Ceased EP1728079A4 (fr)

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